ABSTRACT Diabetes is associated with altered carbohydrate, amino acid, and fatty acid metabolism, contributing to diabetic complications, including diabetic peripheral neuropathy (PN). Diabetic PN affects ~60% of diabetic patients and is characterized by progressive loss of peripheral nerves in a stocking and glove pattern (extremities affected first), with pain and eventual loss of sensation. Despite extensive research over recent decades, the pathogenesis of DN remains unclear, and there is no treatment beyond traditional glucose control, which hardly affects PN development and progression in type 2 diabetes (T2D). Therefore, there is a critical need to determine the specific metabolic mechanisms contributing to the onset and progression of DN in order to identify mechanism-based intervention strategies. Our long-term goal is to meet this need and develop much-needed therapies that impact PN before the onset of disease in order to significantly improve quality of life of diabetic patients. In the current proposal, our objective is to leverage human subjects enrolled in an NIH-funded clinical study at the Investigational Weight Management Clinic (IWMC) at the University of Michigan and a mouse model of high fat diet (HFD)-induced obesity, prediabetes, and PN to identify the mechanisms that contribute to PN pathogenesis. We hypothesize that distinct metabolic alterations occur in obesity, prediabetes, and T2D which induce metabolic reprogramming within the peripheral nerve, altering fuel utilization and ultimately leading to tissue dysfunction. We will test this hypothesis in two aims. First, we will use sensitive and specific mass spectrometry-based metabolomic analysis on plasma from obese, prediabetic human subjects with PN in the IWMC study and from mouse models with PN. Mice will be fed standard diet (SD) or HFD from 5-16 wk of age. We will compare the metabolomic profiles between humans and mice with PN, with the goal of identifying both common and distinct signatures that associate with PN. This cross-species approach will allow us to discover pathogenic metabolomic signatures to act as biomarker(s) of PN in man and mouse, and to identify candidate pathways and molecules whose regulation play crucial roles in the pathogenesis of PN. Second, we will measure changes in mitochondrial function and fuel substrate utilization in peripheral nerve from the mice with PN before and after weight loss. We will use five groups of mice: mice fed SD or HFD from 5-16 wk of age, mice fed a SD or HFD from 5-24 wk, and mice fed a HFD from 5-16 wk then switched to SD from 16-24 wk [HFD- dietary reversal (HFD-DR)]. Notably, HFD-DR mice show significant regression of all PN parameters at 24 wk. These studies will link nerve-specific bioenergetic abnormalities and PN phenotypes to identify candidate bioenergetic pathways whose regulation play crucial roles in the pathogenesis of PN. Together, the proposed studies using obese, prediabetic patients and mice with PN will increase our understanding of how the peripheral nerve adapts to chronic and specific changes in substrate availability beyond excess glucose.